Two Novel lncRNAs Regulate Primordial Germ Cell Development in Zebrafish

Long noncoding RNAs (lncRNAs) are regulatory transcripts in various biological processes. However, the role of lncRNAs in germline development remains poorly understood, especially for fish primordial germ cell (PGC) development. In this study, the lncRNA profile of zebrafish PGC was revealed by single cell RNA-sequencing and bioinformatic prediction. We established the regulation network of lncRNA-mRNA associated with PGC development, from which we identified three novel lncRNAs—lnc172, lnc196, and lnc304—highly expressing in PGCs and gonads. Fluorescent in situ hybridization indicated germline-specific localization of lnc196 and lnc304 in the cytoplasm and nucleus of spermatogonia, spermatocyte, and occyte, and they were co-localized with vasa in the cytoplasm of the spermatogonia. By contrast, lnc172 was localized in the cytoplasm of male germline, myoid cells and ovarian somatic cells. Loss- and gain-of-function experiments demonstrated that knockdown and PGC-specific overexpression of lnc304 as well as universal overexpression of lnc172 significantly disrupted PGC development. In summary, the present study revealed the lncRNA profile of zebrafish PGC and identified two novel lncRNAs associated with PGC development, providing new insights for understanding the regulatory mechanism of PGC development.


Introduction
Primordial germ cells (PGCs), the precursors of germline, segregate at early embryonic development and migrate towards the genital ridge, where they proliferate and differentiate into gonocytes that give rise to gametes [1][2][3]. The migration process imposes significant challenges for PGCs, as along the migratory route PGCs are exposed to various signals that direct differentiation of somatic cell types [4][5][6]. In Drosophila melanogaster, PGCs migrate across the midgut to the mesoderm towards the somatic gonadal precursors [7]. In addition, mouse PGCs migrate from the posterior primitive streak over the endoderm, across the midgut to reach mesoderm, where the primordial gonad develops [8]. Fish PGCs find their way to the genital ridge by a complex migration route through six distinct steps using intermediate targets throughout the embryo [9]. The chemokine-guided cell migration is one of the most important principles of directing PGC migration, conserved in fish and mammals [10]. The G protein-coupled receptor (GPCR)-mediated chemokine attractant is essential for guiding PGC migration towards the genital ridge, such as that trapped in endoderm 1 (TRE1) in Drosophila melanogaster [11] and chemokine (CXC motif) receptor 4b (CXCR4B) in teleost and mammals [10,12].
In lower animals, including teleost fish, PGC development is dependent on molecules that reside within the maternally inherited germ plasm, the non-membrane phase-separated

Fish Care and Maintenance
Tg (kop: EGFP-3 UTR-nanos) zebrafish of AB genetic background were obtained from the China zebrafish resource center (CZRC, Wuhan, China). Zebrafish (Danio rerio) were raised and maintained at 28 • C with 10 h (h) darkness and 14 h light. The embryos were incubated in Ringer's solution in 28 • C incubator (Xutemp, China, Hangzhou). All the procedures with zebrafish were approved by the Ethics Committee of Sun Yat-sen University.

Isolation of PGCs
All the embryos were collected after natural spawning and staged as previously reported [42]. GFP-labelled PGC were isolated from Tg embryos at 5 hpf. The chorions were removed by 0.25% pronase (Roche, Mannheim, Germany) in an embryonic medium as the zebrafish book described (http://zfin.org/zf_info/zfbook/zfbk.html, accessed on 1 January 2022). The de-chorion embryos were disassociated to a single cell in a deyolking buffer (55 mM NaCl, 1.8 mM KCl and 1.25 mM NaHCO 3 ), rinsed twice in a washing buffer (110 mM NaCl, 3.5 mM KCl and 2.7 mM CaCl 2 dissolved in 10 mM Tris/Cl, pH 8.5), and resuspended in nuclease-free PBS. Single GFP-labelled PGC was collected with a capillary tube under a fluorescence microscope (Zesis, Observer Z1, Gottingen, Germany). The collected single PGC was frozen in each tube containing the lysis buffer for single-cell RNA library construction from Annoroad gene technology Co. Ltd. (Beijing, China). A total of 30 PGCs were collected in 30 tubes. After quality checking, 3 of the 30 samples were used for scRNA-seq independently.

scRNA-seq
The collected cells were directly amplified to construct the cDNA library by the Smart-Seq2 method [22] and were sequenced on the Illumina HiSeq 2500 by Annoroad gene technology Co. Ltd. (Beijing, China). The raw data have been submitted to the NCBI SRA Cells 2023, 12, 672 3 of 15 database (PRJNA811725). The raw data were processed with Perl scripts to filter reads with more than 5 adapter-polluted bases, reads with less than 19 accounting for more than 15% and reads with the number of N bases over 5%. Clean reads were mapped to the reference genome danRer10 downloaded from the Ensembl database using Bowtie2 v2.2.3. Gene expression was calculated with fragments per kilobase million mapped fragments (FPKM).

LncRNA Analysis
Known lncRNAs were obtained by blasting to known lncRNA databases (ZFLNC, NONCODE, NCBI, and Ensembl). To obtain putative novel lncRNAs, potential coding transcripts were removed by three types of forecasting software including the coding potential calculator, pfam-scan, and Coding-Non-Coding-Index as previously described [43]. The cis-and trans-target mRNAs of lncRNAs were computationally predicted with the following rules: the potential cis-targets were the protein-coding genes within a 10 k bp window upstream or downstream of the lncRNAs and with a correlation value larger than 0.9; and the trans-targets were predicted on the basis of the mRNA-lncRNA sequence complementarity and the RNA duplex energy. The sequence complementarity was analyzed by BLAST, and the duplex energy was assessed by RNAplex software with the threshold of correlation larger than 0.9 [44]. Gene Ontology enrichment was analyzed using Goatools (https://github.com/tanghaibao/Goatools, accessed on 1 January 2022).

Reverse Transcription and qPCR Analysis
To validate the scRNA transcriptome, strand cDNA libraries of singe PGC were first constructed with a single cell sequence−specific amplification kit (Vazyme, Nanjing, China). The total RNA of adult tissues and embryos were extracted using a TRIzol reagent (Invitrogen, Waltham, MA, USA), and the RNAs were reverse transcribed with GoScript TM reverse transcription mix, random primer (Promega, Madison, WI, USA). qPCR was performed on the Roche Light Cycler 480II (Roche, Rotkreuz, Switzerland). The thermal cycling conditions were as previously described [26]. ef1a and rpl13a were used as reference genes to calculate gene expression with 2 -∆∆Ct method. All the data were represented with mean ± SD from three independent experiments in triplicates. The primers used in this study are listed in Table S1.

Double Color Staining of Immunofluorescence and Fluorescence In Situ Hybridization
The sequence-specific DNA fragments of lnc196, lnc304, and lnc172 were amplified with a pair of primers whose reverse primer contained the core sequence of T7 promoter at the 5 end (primer and lncRNA sequence information in Table S1). Digoxin-labelled antisense RNA probes were synthesized using the DIG-RNA labeling kit (Roche, Mannheim, Germany). The gonads were fixed in 4% PFA at room temperature for 2 h, washed in PBS for 3 × 10 min, and embedded with frozen section compound (Sakura Finetek, Torrance, USA). The samples were sectioned with a thickness of 10 µm. The slides were dried at 37 • C for 30 min, rehydrated, and washed in PBS three times and hybridized with DIG-labeled anti-sense probes at 60 • C for 16 h. After a series of washing steps, the slides were blocked in 2% blocking reagent (Roche, Mannheim, Germany) in MAB buffer (0.1 M Maleic acid and 0.15 M NaCl, pH 7.4) for over 2 h at room temperature and incubated in anti-DIG-POD FAB fragments (Roche, 1:2000 diluted in 1% blocking solution) and anti-vasa antibodies (abcam, ab209710, 1:400) at 4 • C overnight. After washing in PBT and PBS buffer three times, the slides were incubated in TSA™ Plus Fluorescence Systems (Red) (PerkinElmer), FITC-conjugated goat anti-rabbit IgG (H + L), and DAPI (1 µg/mL) for 1 h. The images were acquired with a Leica Stellaris 5 confocal microscope (Leica, Mannheim, Germany).

lncRNA Synthesis
For lncRNA synthesis, full-length DNA fragments of lncRNA were inserted into the pCS2+ vector with poly A terminator removed by double digestion of Xho I and Not I enzymes. For PGC-specific overexpression of lncRNAs, the 3 UTR of nos3 was fused to

Knockdown and Overexpression
Sequence-specific antisense oligonucleotides (ASOs) were synthesized (RiboBio, Guangzhou, China). Different dosages of lnc196, lnc304, and lnc172 ASOs and control ASO were injected into 1-cell stage Tg (kop: EGFP-UTR-nanos3) embryos, respectively. The injected embryos were collected for qPCR analysis and tracing PGC development. For overexpression, 200 pg lncRNAs were injected to 1-cell stage embryos, and embryos injected with 200 pg rfp mRNA were used as control. Rescue experiments were performed by co-injection of 200 pg lncRNAs and 10 pM corresponding ASOs into 1-cell stage Tg embryos. All the experiments were performed in more than three independent experiments in triplicate. Each independent experiment was performed in triplicate.

PGC Phenotype Observation
The PGC development was examined at 24 hpf under the stereo fluorescence microscope (SMZ800N, Nikon, Kyoto, Japan). The standard for the phenotype of PGC mislocalization was described as in the previous report [26]. The phenotypes were determined by four independent experiments with four groups of embryos (30-50 embryos per group).

Statistics Analysis
The statistics were calculated and analyzed with SPSS version 20. The qPCR results were analyzed by Student's t-test, and the phenotypes in the different experimental groups were analyzed by one-way ANOVA. p < 0.5 was considered a statistically significant difference.

Identification and Characterization of lncRNA in Zebrafish PGC by scRNA-seq
Single PGC was collected with a capillary tube from the embryos of a PGC-specific transgenic line (Tg kop: egfp−UTR−nos3) at 5 h post fertilization (hpf) as previously described [45] ( Figure S1). The collected PGCs were independently divided into three groups for scRNA-seq by Illumina platform. The transcriptome data obtained 274,016,310 clean reads, which were annotated to 23,099 genes. In the top 25 genes, there were five known PGC marker genes including ca15b, nanos3, dnd1, gra, and tdrd7a and two mitochondria components, mt-nd4 and mt-nd5 ( Figure 1A). Gene ontology analysis showed that the top 100 genes were enriched in the components and biological processes that were matched with the intrinsic features of PGC, including cytoplasmic ribonucleoprotein granule, nonmembrane-bounded organelle, germ plasm, and the biological process associated with RNA/mRNA metabolism, stabilization, chromatin assembly, and post-transcriptional regulation of gene expression ( Figure 1B,C). The top genes and their enriched functional signaling pathways demonstrated that the cells used for scRNA-seq are PGCs.

Screening Out lncRNA Candidates Associated with PGC Development in Zebrafish
As regulatory transcripts, lncRNAs themselves have limited potential to code protein products; therefore, they usually interact with other coding genes to modulate the biological process. To characterize the role of lncRNA in PGC development, cis-and trans-targets of the novel lncRNAs have been predicted. The results revealed that 147 lncRNAs were potentially targeted to 112 mRNAs, forming 1352 pairs of lncRNA and mRNA (Table S2). To facilitate the search for lncRNA associated with PGC development, we focused on the known important PGC factors within predicted targets and their lncRNAs to construct a regulation network consisting of 69 lncRNAs and 18 known PGC development-associated genes including tudor domain containing (tdrd) family, piwi-like RNA-mediated gene silencing 1 (piwil1), chemokine (C-X-C motif) receptor 7 (cxcr7), gamma-glutamyltransferase 1a (ggt1), regulator of G protein signaling 14a (rgs14a), and nanos homolog 1 (nos1) (Figure 3, Table S3). From the network, we selected the top nine lncRNAs to analyze their expression in zebrafish PGCs and soma. The qPCR results showed that lnc196, lnc304, lnc172, lnc370, lnc279, and lnc181 were PGC-biased, whereas lnc114, lnc308, and lnc345 were soma-biased ( Figure 4A). Because lnc196, lnc304, and lnc172 were the most PGC-enriched lncRNAs, the spatial and temporal expression and localization were further characterized in adult tissues and embryos. In adult tissues, lnc196, lnc304, and lnc172 were all enriched in the testes ( Figure 4B-D). During embryonic development, lnc196 existed in the unfertilized egg and increased in tandem with the embryonic development; lnc304 was expressed from the shield stage; the maternally-expressed lnc172 quickly decreased and was almost undetectable at 30% epiboly ( Figure 4E-G).

Screening Out lncRNA Candidates Associated with PGC Development in Zebrafish
As regulatory transcripts, lncRNAs themselves have limited potential to code protein products; therefore, they usually interact with other coding genes to modulate the biological process. To characterize the role of lncRNA in PGC development, cis-and trans-targets of the novel lncRNAs have been predicted. The results revealed that 147 lncRNAs were potentially targeted to 112 mRNAs, forming 1352 pairs of lncRNA and mRNA (Table  S2). To facilitate the search for lncRNA associated with PGC development, we focused on the known important PGC factors within predicted targets and their lncRNAs to construct a regulation network consisting of 69 lncRNAs and 18 known PGC development-associated genes including tudor domain containing (tdrd) family, piwi-like RNA-mediated gene silencing 1 (piwil1), chemokine (C-X-C motif) receptor 7 (cxcr7), gamma-glutamyltransferase 1a (ggt1), regulator of G protein signaling 14a (rgs14a), and nanos homolog 1 (nos1) (Figure 3, Table S3). From the network, we selected the top nine lncRNAs to analyze their expression in zebrafish PGCs and soma. The qPCR results showed that lnc196, lnc304, lnc172, lnc370, lnc279, and lnc181 were PGC-biased, whereas lnc114, lnc308, and lnc345 were soma-biased ( Figure 4A). Because lnc196, lnc304, and lnc172 were the most PGC-enriched lncRNAs, the spatial and temporal expression and localization were further characterized in adult tissues and embryos. In adult tissues, lnc196, lnc304, and lnc172 were all enriched in the testes ( Figure 4B-D). During embryonic development, lnc196 existed in the unfertilized egg and increased in tandem with the embryonic development; lnc304 was expressed from the shield stage; the maternally-expressed lnc172 quickly decreased and was almost undetectable at 30% epiboly ( Figure 4E-G).   The roles of lncRNA are closely associated with subcellular localization. we characterized the subcellular localization of lnc172, lnc196, and lnc304 in zeb ads by fluorescent in situ hybridization (FISH). In mature testes, lnc196 and ln localized in the nucleus and cytoplasm of spermatogonia and spermatocytes signals gradually weakened from the spermatogonia over primary spermatoc ondary spermatocyte ( Figure 5A-F). Notably, both lnc196 and lnc304 were pa localized with vasa in the cytoplasm of the spermatogonia (the magnified imag C&F) ( Figure 5). By contrast, lnc172 was localized in the cytoplasm of the sperm spermatocyte, and myoid cell ( Figure 5G-H). In a mature ovary, lnc196 and ln mainly localized in the nucleus of the pre-vitellogenesis oocyte ( Figure 6A-F), was localized in the cytoplasm of the ovarian somatic cell (Figure 6G-H). The The roles of lncRNA are closely associated with subcellular localization. Therefore, we characterized the subcellular localization of lnc172, lnc196, and lnc304 in zebrafish gonads by fluorescent in situ hybridization (FISH). In mature testes, lnc196 and lnc304 were localized in the nucleus and cytoplasm of spermatogonia and spermatocytes, and their signals gradually weakened from the spermatogonia over primary spermatocyte to secondary spermatocyte ( Figure 5A-F). Notably, both lnc196 and lnc304 were partially colocalized with vasa in the cytoplasm of the spermatogonia (the magnified images in panel C&F) ( Figure 5). By contrast, lnc172 was localized in the cytoplasm of the spermatogonia, spermatocyte, and myoid cell ( Figure 5G-H). In a mature ovary, lnc196 and lnc304 were mainly localized in the nucleus of the pre-vitellogenesis oocyte ( Figure 6A-F), and lnc172 was localized in the cytoplasm of the ovarian somatic cell ( Figure 6G-H). The germlineenriched expression patterns suggested that lnc172, lnc196, and lnc304 might regulate PGC development.

Disruption of lnc172 and lnc304 Affected PGC Development
To investigate the potential role of lnc172, lnc196, and lnc304 in PGC development, we performed loss of-and gain-of-function experiments by injection of sequence-specific antisense oligonucleotides (ASO) and lncRNAs, respectively. The qPCR results indicated that injection of ASOs significantly decreased endogenous lncRNAs (Figures 7A and S2A). Next, we examined the knockdown of lncRNAs affected PGC development. Compared to the control ASO-injected embryos, PGC developed normally in lnc172 or lnc196. The ASOinjected embryos are shown at 24 h post fertilization (hpf) in Figure S2. There was significant PGC mislocalization in the lnc304 ASO-injected embryos ( Figures 7B-E, S2B, C, F, and G). In the lnc304 ASO-injected embryos, the percentage of embryos with mislocalized PGC and the average number of mislocalized PGC in each embryo increased to 26.56% and 4.88, respectively ( Figure 7K,L). We also investigated whether overexpression of the lncRNAs affected PGC development. Compared to rfp mRNA-injected embryos, we

Disruption of lnc172 and lnc304 Affected PGC Development
To investigate the potential role of lnc172, lnc196, and lnc304 in PGC development, we performed loss of-and gain-of-function experiments by injection of sequence-specific antisense oligonucleotides (ASO) and lncRNAs, respectively. The qPCR results indicated that injection of ASOs significantly decreased endogenous lncRNAs ( Figure 7A and Figure  S2A). Next, we examined the knockdown of lncRNAs affected PGC development. Compared to the control ASO-injected embryos, PGC developed normally in lnc172 or lnc196. The ASO-injected embryos are shown at 24 h post fertilization (hpf) in Figure S2. There was significant PGC mislocalization in the lnc304 ASO-injected embryos ( Figure 7B-E and Figure S2B,C,F,G). In the lnc304 ASO-injected embryos, the percentage of embryos with mislocalized PGC and the average number of mislocalized PGC in each embryo increased to 26.56% and 4.88, respectively ( Figure 7K,L). We also investigated whether overexpression of the lncRNAs affected PGC development. Compared to rfp mRNA-injected embryos, we observed mislocalized PGCs in the lnc172and lnc304-injected embryos ( Figure 7F-H) but not in the lnc196-injected embryos ( Figure S2D-G). The percentage of embryos with mislocalized PGCs and the average number of mislocalized PGCs in each embryo increased to 36.77% and 5.17 and 50.37% and 6.31 in the lnc304 and lnc172 injected embryos, respectively ( Figure 7K-N). Co-injection of the lnc304 and lnc172 ASOs with corresponding lncRNAs significantly decreased the percentage of embryos with mislocalized PGCs and the average number of mislocalized PGCs (Figure 7I-N). These loss of-and gain of-function results indicated that lnc172 and lnc304 played important roles in PGC development. observed mislocalized PGCs in the lnc172-and lnc304-injected embryos ( Figure 7F-H) but not in the lnc196-injected embryos ( Figure S2D-G). The percentage of embryos with mislocalized PGCs and the average number of mislocalized PGCs in each embryo increased to 36.77% and 5.17 and 50.37% and 6.31 in the lnc304 and lnc172 injected embryos, respectively ( Figure 7K-N). Co-injection of the lnc304 and lnc172 ASOs with corresponding lncRNAs significantly decreased the percentage of embryos with mislocalized PGCs and the average number of mislocalized PGCs ( Figure 7I-N). These loss of-and gain of-function results indicated that lnc172 and lnc304 played important roles in PGC development.  Results were representative of more than three independent experiments in triplicate. ef1a and rpl13a were used as internal controls to normalize gene expression levels with 2 -∆∆Ct method. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. Scale bar, 100 µm.

lnc172 and lnc304 Regulate PGC Development in Different Ways
Universal overexpression of lnc172and lnc304-mediated developmental abnormalities might be caused by lncRNA overexpression in soma, PGC, or both. To clarify this, we fused the lncRNAs with 3 untranslated region of nos3 (the lnc304-nos3 3 utr and lnc172-nos3 3 utr) that will be specifically overexpressed in the PGCs because of somatic clearance by the miR-430 from gastrulation [46]. Compared to the embryos injected with lnc304 ( Figure 8A-C), the percentage of embryos with mislocalized PGC and the average number of mislocalized PGC in each embryo significantly increased to 64.71% and 7.29 in the lnc304-nos3 3 utr injected embryos ( Figure 8D,G). By contrast, we found normal PGC development in the lnc172-nos3-3 utr-injected embryos ( Figure 8E,F,H), suggesting that the developmental defects of PGC in the lnc172 injected embryos required somatic overexpression of lnc172.
These results indicate different ways lnc304 and lnc172 function in the regulation of PGC development: PGC-specific overexpression of lnc304 and soma overexpression of lnc172 disrupted the PGC development.

lnc172 and lnc304 Regulate PGC Development in Different Ways
Universal overexpression of lnc172-and lnc304-mediated developmental abnormalities might be caused by lncRNA overexpression in soma, PGC, or both. To clarify this, we fused the lncRNAs with 3′ untranslated region of nos3 (the lnc304-nos3 3′utr and lnc172-nos3 3′utr) that will be specifically overexpressed in the PGCs because of somatic clearance by the miR-430 from gastrulation [46]. Compared to the embryos injected with lnc304 (Figure 8A-C), the percentage of embryos with mislocalized PGC and the average number of mislocalized PGC in each embryo significantly increased to 64.71% and 7.29 in the lnc304-nos3 3′utr injected embryos ( Figure 8D,G). By contrast, we found normal PGC development in the lnc172-nos3-3′utr-injected embryos ( Figure 8E,F,H), suggesting that the developmental defects of PGC in the lnc172 injected embryos required somatic overexpression of lnc172. These results indicate different ways lnc304 and lnc172 function in the regulation of PGC development: PGC-specific overexpression of lnc304 and soma overexpression of lnc172 disrupted the PGC development.

Discussion
In the present study, we revealed lncRNA profiles of zebrafish PGC, established the regulation network of lncRNA-mRNA associated with PGC, and found two novel lncRNAs lnc172 and lnc304, which play important roles in PGC development. To the best of our knowledge, it is the first report about the function of lncRNAs in the PGC development of fish.
Zebrafish PGCs specify at ~3 hpf, polarize at ~4.5 hpf, initiate to migrate at ~5 hpf, and reach to the genital ridge and stop migration at 24 hpf [47]. Genome-wide transcriptional silence is required for PGC specification and migration in fruit flies and zebrafish [32,48]. A recent study reports that migratory PGCs at 6 and 11 hpf shared a similar gene expression pattern [41]. In this study, we revealed the lncRNA profiles of zebrafish PGC

Discussion
In the present study, we revealed lncRNA profiles of zebrafish PGC, established the regulation network of lncRNA-mRNA associated with PGC, and found two novel lncRNAs lnc172 and lnc304, which play important roles in PGC development. To the best of our knowledge, it is the first report about the function of lncRNAs in the PGC development of fish.
Zebrafish PGCs specify at~3 hpf, polarize at~4.5 hpf, initiate to migrate at~5 hpf, and reach to the genital ridge and stop migration at 24 hpf [47]. Genome-wide transcriptional silence is required for PGC specification and migration in fruit flies and zebrafish [32,48]. A recent study reports that migratory PGCs at 6 and 11 hpf shared a similar gene expression pattern [41]. In this study, we revealed the lncRNA profiles of zebrafish PGC at 5 hpf when PGCs initiate migration, providing useful information for studying the role of lncRNA in PGC development.
In the lncRNA-mRNA regulation network, there were 12 known RNPs including piwil2, dazap1/2, tdrd1/2/3/5/6/7a/12/15, and tdrdkh. Piwil2 interacts with the piRNAs, which is critical for maintaining transposon silence in zebrafish PGC [24]. The germlinespecific family TDRDs are indispensable for gametogenesis and genome stability via the piRNA pathway [49,50]. DAZ families are required for PGC migration, as is commitment to germline fate by regulation of RNA stability and translation [23,51,52]. The regulation network suggested that these germline-specific RBPs also interact with lncRNAs. Besides, six lncRNAs were predicted to target with three PGC migration factors including rgs14a, cxcr7a, and hmgcr [10,19,53,54], suggesting that they might be involved in the regulation of PGC migration. From the network, we identified six PGC-based novel lncRNAs including lnc196, lnc304, and lnc172, which were predicted to regulate piwil2, nr0b1, and tdrd12, respectively. However, whether other lncRNAs involved in the regulation network might be associated with PGC development and the interaction among these lncRNAs and PGCassociated genes is a question that should be investigated in the future.
Generally, nucleus-localized lncRNAs are associated with the regulation of chromatin topology and neighboring genes [55][56][57], and cytoplasm-localized lncRNAs act as scaffolds of proteins and RNA to regulate maintenance and decay [58,59]. In this paper, we investigated the subcellular localization of lnc172, lnc196, and lnc304 in embryos and gonads and observed strong signals in the gonads ( Figures 5 and 6), but we failed to find a specific signal in PGCs (data not shown). We speculated that the reason might that in situ hybridization is not sensitive enough to detect low levels of lncRNA in PGC in comparison with qPCR analysis. In the testes, lnc196 and lnc304 were co-localized with vasa in the spermatogonia, and the signals weakened from the spermatogonia to the secondary spermatocyte, which is consistent with the expression pattern of fish germ plasm components [60][61][62]. The nucleus localization of lnc196 and lnc304 in male germline and pre-vitellogenic oocyte indicated that they might be associated with chromatin structure and transcriptional regulation. lnc172 was a highly expressed myoid cell and ovarian somatic cell, which are the gonadal somatic cells that play important role in germline self-renewal, differentiation, and maintenance via sexual hormones synthesis and response [63][64][65]. In summary, the subcellular localization suggests that lnc172, lnc196, and lnc304 might play important roles during gametogenesis.
Zebrafish PGCs employ "amoeboid migration" to migrate towards the genital ridge, which is fine-tuned by intrinsic factors and the surrounding niche [5,66]. The onset of PGC migration accompanies the decrease in E-cadherin, whose expression is controlled by Rgs14a, a scaffolding protein in PGCs [19]. The bleb formation and motility of PGC require high myosin-based contractility and cortex properties [67]. Soma-derived chemokine signaling is indispensable for the guiding of PGC to arrive at their specific intermediate and final targets. Disruption of factors involved in chemokine signaling such as sdf1, cxcr4, and cxcr7 destroy directional migration, leading to PGCs dispersed in the trunk [68][69][70]. In the present study, we found that knockdown or PGC-specific overexpression of lnc304 and universal overexpression are probably due to the somatic overexpression of lnc172, resulting in PGC scatted in the tail, somite, eyes, and brain (Figures 7 and 8), similar to the phenotypes observed in cxcr4 morpholino-injected embryos. These results demonstrated that proper expression of lncRNAs in soma and PGC is important for directing PGC migration towards the genital ridge. However, the detail process and mechanism of how lnc172 and lnc304 regulate PGC need to be further investigated.
In summary, the present study provided lncRNA profiles of zebrafish PGC and identified two novel lncRNA critical in directing PGC migration, which is helpful for understanding the role of lncRNA in PGC development.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12040672/s1, Figure S1: The procedure of PGC isolation for scRNA-seq; Figure S2: PGC development in embryos injected with lnc196 RNA or lnc196 ASO; Table S1: The sequence information of the primers and lncRNAs; Table S2: The predicted cis-and trans-targets of novel lncRNAs; Table S3: The sequence of lncRNA and ensemble ID of corresponding target RNA in network.